Difference in Risk Factors for Breast Cancer by ER Status in an Indigenous African Population.

Introduction. Breast cancer is the commonest cancer among women globally. In Uganda, it is on the rise, projected at a 4.5% annual ASR increase (age standardized incidence rate). The reasons for this steep increase are not fully established. In the recent past, gene profiling in tumor tissues suggests that breast cancers are divided into subtypes dependent on the presence or absence of oestrogen receptor, progesterone, and human epidermal growth factor receptor 2 (HER 2). These subtypes do have distinctive clinical outcomes and perhaps risk factors from past studies. There is paucity of data on hormonal receptor status and the traditionally known risk factors in sub-Saharan Africa. The purpose of this study therefore was to establish the differences between ER status and the traditionally known risk factors for breast cancer in Uganda. Methods. An observational analytical hospital, based study, carried out at Makerere University, College of Health Sciences. Formalin fixed and paraffin imbedded sections were prepared for haemotoxylin and eosin (H&E) stains and immunohistochemistry (IHC). Ethical approval was obtained. Results. A total of 113 women were recruited. Mean age was 45 years (SD14). There were no significant differences in selected risk factors (setting, age, contraceptive use, parity, breast feeding, or menarche) by ER status although ER negative tumors had significantly higher grade tumors (by a factor of two) compared to ER positive tumors. Conclusion. There were no significant differences among risk factors by ER status contrary to what several other studies suggest. The manifestation of breast cancer in Africa warrants further extensive inquiry.

Role of human and mouse HspB1 in metastasis.

Heat shock proteins (HSP) are a group of physiologically-essential, highly-conserved proteins that are induced by heat shock, as well as by other environmental and pathophysiological stressors. The twentyseven kDa heat shock protein (Hsp27; HspB1) is highly expressed in tumor tissues of patients diagnosed with cancer and expression levels correlate with poor prognosis. HspB1 plays a dual role in cancer and promotes both cancer development by suppressing host anti-cancer response, such as apoptosis and senescence, and facilitates the enhanced expression of metastastic genes. HspB1-mediated protection from tumor cell apoptosis induced by chemotherapeutic drugs occurs through several mechanisms, including decreased production of reactive oxygen species, restoration of protein homeostasis and promotion of cell survival by protein folding, stabilization of actin-cytoskeleton, delayed release of cytochrome c from mitochondria and inhibition of activation of caspase-3. High levels of HSP expression affect tumor susceptibility to adjuvant cancer treatments, including chemotherapy, hyperthermia, and radiation. This review highlights the most recent findings and role of HspB1 in metastasis.

Enteroaggregative Escherichia coli: An Emerging Enteric Food Borne Pathogen.

Enteroaggregative Escherichia coli (EAEC) are quite heterogeneous category of an emerging enteric pathogen associated with cases of acute or persistent diarrhea worldwide in children and adults, and over the past decade has received increasing attention as a cause of watery diarrhea, which is often persistent. EAEC infection is an important cause of diarrhea in outbreak and non-outbreak settings in developing and developed countries. Recently, EAEC has been implicated in the development of irritable bowel syndrome, but this remains to be confirmed. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence (AA) to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa with a "stacked-brick" adherence phenotype, which is related to the presence of a 60 MDa plasmid (pAA). At the molecular level, strains demonstrating the aggregative phenotype are quite heterogeneous; several virulence factors are detected by polymerase chain reaction; however, none exhibited 100% specificity. Although several studies have identified specific virulence factor(s) unique to EAEC, the mechanism by which EAEC exerts its pathogenesis is, thus, far unknown. The present review updates the current knowledge on the epidemiology, chronic complications, detection, virulence factors, and treatment of EAEC, an emerging enteric food borne pathogen.

The effect of stress-inducible extracellular Hsp72 on human neutrophil chemotaxis: a role during acute intense exercise.

We studied the physiological role of the 72 kDa extracellular heat shock protein (Hsp72, a stress-inducible protein) in modulating neutrophil chemotaxis during a single bout of intense exercise performed by sedentary women, together with various cell mechanisms potentially involved in the modulation. For each volunteer, we evaluated neutrophil chemotaxis and serum Hsp72 concentration before and immediately after a single bout of exercise (1 h on a cycle ergometer at 70% VO(2) max), and 24 h later. Both parameters were found to be stimulated by the exercise, and had returned to basal values 24 h later. In vitro, there was a dose-dependent increase in chemotaxis when neutrophils were incubated both with physiological Hsp72 concentrations and with a 100 x greater concentration. The chemotaxis was greater when the neutrophils were incubated with the post-exercise Hsp72 concentration than with the basal concentration, suggesting a physiological role for this protein in the context of the stimulation of neutrophil chemotaxis by intense exercise. The 100 x Hsp72 concentration stimulated chemotaxis even more strongly. In addition, Hsp72 was found to have chemoattractant and chemokinetic effects on the neutrophils at physiological concentrations, with these effects being significantly greater with the post-exercise than with the basal Hsp72 concentration. The Hsp72-induced stimulation of neutrophil chemotaxis disappeared when the toll-like receptor 2 (TLR-2) was blocked, and phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and nuclear transcription factor kappa B (NF-kappaB) were also found to be involved in the signaling process. No changes were observed, however, in neutrophil intracellular calcium levels in response to Hsp72. In conclusion, physiological concentrations of the stress protein Hsp72 stimulate human neutrophil chemotaxis through TLR-2 with its cofactor CD14, involving ERK, NF-kappaB, and PI3K, but not iCa(2 + ), as intracellular messengers. In addition, Hsp72 seems to participate in the stimulation of chemotaxis induced by a single bout of intense exercise performed by sedentary women.

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

Targeting HSP70-induced thermotolerance for design of thermal sensitizers.

Thermal therapy has been shown to be an extremely powerful anti-cancer agent and a potent radiation sensitizer. However, the full potential of thermal therapy is hindered by a number of considerations including highly conserved heat resistance pathways in tumour cells and inhomogeneous heating of deep-seated tumours due to energy deposition and perfusion issues. This report reviews recent progress in the development of hyperthermia sensitizing drugs designed to specifically amplify the effects of hyperthermia. Such agents might be particularly useful in situations where heating is not adequate for the full biological effect or is not homogeneously delivered to tumours. The particular pathway concentrated on is thermotolerance, a complex, inducible cellular response that leads to heat resistance. This paper will concentrate on the molecular pathways of thermotolerance induction for designing inhibitors of heat resistance/thermal sensitizers, which may allow the full potential of thermal therapy to be utilized.

Cyclooxygenase inhibitors are potent sensitizers of prostate tumours to hyperthermia and radiation.

It has previously been demonstrated that hyperthermia can activate prostaglandin synthesis and that prostaglandins are protective against hyperthermia. This study examined the use of inhibitors of prostaglandin synthesis on the response of prostate tumours to hyperthermia. The non-steroidal anti-inflammatory drugs (NSAID) ibuprofen and sulindac, known cyclooxygenase inhibitors that inhibit prostaglandin production, were effective hyperthermia sensitizers and augmented growth delay of DU-145 and PC-3 prostate tumours to combined radiation and hyperthermia treatment protocols. Pre-treatment of mice with ibuprofen and sulindac at hyperthermia sensitizing doses resulted in significant (p < 0.01) inhibition of hyperthemia-induced serum prostaglandin E2. These findings indicate that NSAID may have both sensitizing effects on prostate tumour growth and may function by inhibiting prostaglandin synthesis.

Effects of the flavonoid drug quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo.

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.

Role of calcium activated kinases and phosphatases in heat shock factor-1 activation.

HSF-1 is regulated at multiple molecular levels through intra- and intermolecular protein-protein interactions as well as by post-translational modification through phosphorylation. We have found that elevating intracellular calcium ion levels by exposure to the ionophore A23187 or thapsigargin inhibits the conversion of HSF-1 from a latent cytoplasmic form to its nuclear/DNA binding form. To examine a role for calcium/calmodulin regulated enzymes in this process, we examined the ability of specific inhibitors to abrogate the effects of calcium elevation. While the inhibitor of calmodulin dependent kinase II, KCN62 enhanced activation of HSF-1 during heat shock, it failed to block the inhibitory effects of calcium increase. By contrast, the immunosuppresant drugs cyclosporin A and FK506 abolished the effects of calcium elevation on HSF-1 activation. As the biological effects of the drugs are effected through inhibition of the calcium/calmodulin regulated phosphatase calcineurin, this suggests a role for calcineurin in antagonizing HSF-1 activity. The experiments suggest the existence of phosphorylated residue(s) in HSF-1 important in one or more of the processes that lead to activation (trimerization, nuclear localization, DNA binding) and which becomes dephosphorylated due to the activation of a calcium/calmodulin/calcineurin complex.

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography.

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.

HSP70 peptidembearing and peptide-negative preparations act as chaperokines.

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca2+]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-kappaB activity and subsequent augmentation of proinflammatory cytokine TNF-alpha, IL-1beta, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

HSP70 and heat shock factor 1 cooperate to repress Ras-induced transcriptional activation of the c-fos gene.

Heat shock protein 70 (HSP70) is a molecular chaperone involved in protein folding and resistance to the deleterious effects of stress. Here we show that HSP70 suppresses transcription of c-fos, an early response gene that is a key component of the ubiquitous AP-1 transcription factor complex. HSP70 repressed Ras-induced c-fos transcription only in the presence of functional heat shock factor1 (HSF1). This suggests that HSP70 functions as a corepressor with HSF1 to inhibit c-fos gene transcription. Therefore, besides its known function in the stress response, HSP70 also has the property of a corepressor and combines with HSF1 to antagonize Fos expression and may thus impact multiple aspects of cell regulation.

RSK2 represses HSF1 activation during heat shock.

Heat shock transcription factor 1(HSF1) activation is a multistep process. The conversion of a latent cytoplasmic form to a nuclear, DNA binding state appears to be activated by nonsteroidal anti-inflammatory drugs. In previous studies, we showed that HSF 1 is phosphorylated by the protein kinase RSK2 in vitro and that this effect is inhibited by nonsteroidal anti-inflammatory drugs at the concentration that leads to the activation of HSF1 in vivo (Stevenson et al 1999). In the present study, using cells from a patient with Coffin-Lowry syndrome (deficient in RSK2), we demonstrate that RSK2 slightly represses activation of HSF1 in vivo at 37 degrees C. In Coffin-Lowry syndrome cells, HSF1-HSE DNA binding activity after treatment with sodium salicylate was slightly higher than that in untreated cells, indicating that although RSK2 is involved in HSF1 regulation, it is not the unique protein kinase that suppresses HSF1-HSE binding activity at 37 degrees C. However, heat shock treatment resulted in significantly higher HSF1-HSE binding activity in Coffin-Lowry syndrome cells as compared with normal controls, suggesting that RSK2 represses HSF1-HSE binding activity during heat shock.

Salicylic acid and aspirin inhibit the activity of RSK2 kinase and repress RSK2-dependent transcription of cyclic AMP response element binding protein- and NF-kappa B-responsive genes.

Sodium salicylate (NaSal) and other nonsteroidal anti-inflammatory drugs (NSAIDs) coordinately inhibit the activity of NF-kappa B, activate heat shock transcription factor 1 and suppress cytokine gene expression in activated monocytes and macrophages. Because our preliminary studies indicated that these effects could be mimicked by inhibitors of signal transduction, we have studied the effects of NSAIDs on signaling molecules potentially downstream of LPS receptors in activated macrophages. Our findings indicate that ribosomal S6 kinase 2 (RSK2), a 90-kDa ribosomal S6 kinase with a critical role as an effector of the RAS-mitogen-activated protein kinase pathway and a regulator of immediate early gene transcription is a target for inhibition by the NSAIDs. NSAIDs inhibited the activity of purified RSK2 kinase in vitro and of RSK2 in mammalian cells and suppressed the phosphorylation of RSK2 substrates cAMP response element binding protein (CREB) and I-kappa B alpha in vivo. Additionally, NaSal inhibited the phosphorylation by RSK2 of CREB and I-kappa B alpha on residues crucial for their transcriptional activity in vivo and thus repressed CREB and NF-kappa B-dependent transcription. These experiments suggest that RSK2 is a target for NSAIDs in the inhibition of monocyte-specific gene expression and indicate the importance of RSK2 and related kinases in cell regulation, indicating a new area for anti-inflammatory drug discovery.

Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4.

In vivo inoculation of specific antibody is an accepted protocol for elimination of specific cell populations. Except for anti-CD3 and anti-CD4, it is not known if the depleted cells are eliminated by signalling through the target molecule or through a more non-specific mechanism. C57BL/6 mice were inoculated with anti-natural killer (NK1.1) monoclonal antibody (mAb). Thereafter spleen cells were harvested, stained for both surface and intracellular markers, and analysed by flow cytometry. As early as 2 hr post inoculation, NK cells were signalled to become apoptotic while signalling through the NK1.1 molecule activated NK1.1+ T-cell receptor (TCR)+ (NK T) cells to increase in number, and produce interleukin-4 (IL-4). Anti NK1.1 mAb was less efficient at signalling apoptosis in NK cells when NK T-cell deficient [beta 2-microglobulin beta 2m-deficient] mice were used compared with wild type mice. Efficient apoptotic signalling was restored when beta 2m-deficient mice were reconstituted with NK T cells. NK-specific antibody best signals the apoptotic process in susceptible NK cells when resistant NK T cells are present, activated, and secrete IL-4.

Tumor suppressive action of indomethacin is NK-cell-independent.

This study was undertaken to determine whether NK-cells constitute a necessary mediator for the suppression of tumor growth by indomethacin. C57Bl mice with a methylcholantrene (MCG 101) tumor were studied. Indomethacin treatment was provided by daily subcutaneous injections (1 microgram/g body weight). NK-cells were depleted by treatment with a monoclonal antibody to NK1.1. Consecutive indomethacin injections prolonged survival in tumor bearing animals. Indomethacin was equally effective in animals with intact NK-cells as in NK-cell-depleted animals. Further, the MCG cells were apparently insensitive to the lytic activity of NK-cells in vivo. Thus, the clearance of intravenously injected MCG cells from lungs was not affected by depletion of NK-cells in vivo; in contrast, the corresponding clearance of NK-cell-sensitive YAC-1 lymphoma cells was strikingly reduced by the depletion of NK-cells. Our data suggest that NK cells are not a necessary mediator for the suppression of tumor growth by indomethacin.

Duration and mechanisms of the increased natural cytotoxicity seen after chronic voluntary exercise in rats.

We have recently shown that in vivo natural cytotoxicity is enhanced after chronic exercise in spontaneously hypertensive rats (SHRs). In the present report, we have studied the duration of this augmentation and some possible mechanisms involved. Exercise consisted of voluntary running for 4-5 weeks, with the running distance ranging from 2.7-15.6 km day(-1) during the last week of running. In vivo cytotoxicity was measured as clearance of injected 51Cr-labelled YAC-1 lymphoma cells from the lungs. The in vivo natural cytotoxicity was increased in running SHRs, and also in SHRs that had their running wheel locked for 24 and 48 h prior to the experiment, and was still present after 96 h. The enhancement of in vivo cytotoxicity after 5 weeks of exercise was abolished after an acute injection of the beta-adrenergic receptor antagonist timolol (0.5 mg kg(-1) i.v.), indicating that catecholamines are involved in this augmentation. Interestingly, 24 h after the last exercise bout, the increased natural cytotoxicity could be blocked by timolol. The opioid receptor antagonist naloxone given subcutaneously for 7 days by osmotic pumps (6 mg kg(-1) h(-1)) could not reverse the increased in vivo cytotoxicity seen in the running SHRs, suggesting that opioid receptor mechanisms are not involved, or at least not the naloxone-sensitive mu-receptor. Natural immunity was not influenced by the histamine H2 receptor antagonist ranitidine, either in controls or in runners, indicating that the natural killer cell-regulatory effect of histamine is not present in SHRs and does not seem to be involved in the exercise-induced changes in natural immune function. We conclude that the augmentation of in vivo natural cytotoxicity after voluntary chronic exercise in rats is long-lasting and that the augmentation is partly mediated by beta-adrenergic receptors.

Acute mental stress but not enforced muscle activity transiently increases natural cytotoxicity in spontaneously hypertensive rats.

The influence of acute mental stress and the effect of electrically induced skeletal muscle contractions on natural cytotoxicity in vivo was investigated in spontaneously hypertensive rats Natural cytotoxicity in vivo was measured as the clearance of injected 51Cr-labelled YAC-1 lymphoma cells from the lungs, which are specifically lysed by natural killer cells. The mental stress consisted of an air jet directed towards the animals in their cage for 25 min. During the mental stress there was a significant increase in natural cytotoxicity. Thus, retained radioactivity in the lungs was decreased to 74 +/- 6% of the control levels which was set to 100% (P < 0.01). This augmentation of YAC-1-cell clearance could be blocked with the beta-adrenergic receptor antagonist Timolol. Two hours after termination of the air stress, in vivo cytotoxicity had returned to control levels. In contrast, acute physical stress, consisting of electrically induced muscle contractions for 60 min, had no significant effects on in vivo cytotoxicity, either during the stimulation or 1, 2 or 24 h after the stimulation. Further, significantly increased plasma levels of adrenaline were seen after the air jet stress, but not after muscle stimulation. There were no significant changes in plasma noradrenaline levels either after air stress or muscle stimulation. These results indicate that changes in in vivo cytotoxicity after mild mental stress are dependent on increased plasma catecholamine levels while acute physical stress without changes in catecholamine levels, does not influence in vivo cytotoxicity.